Heterotrophic Nature of the Cell-Free Protein-Synthesizing System from the Strict Chemolithotroph, Thiobacillus thiooxidans

A cell-free protein-synthesizing system prepared from the strict chemolithotroph, Thiobacillus thiooxidans, was similar to that of heterotrophs. The poly-U directed system had a temperature optimum of 37 C, but in the presence of spermidine (3 mM) the optimum shifted to 45 C. Although growth of the chemolithotroph occurs only in acid conditions, the pH optimum for the cell-free system was pH 7.2. The endogenous-directed activity in the presence or absence of spermidine was maximal at pH 7.8. Spermidine had a stimulatory effect; however, this effect was dependent on the magnesium and tris(hydroxymethyl)aminomethane (Tris) concentrations. At low Tris concentrations (10 mM), spermidine (3 to 5 mM) could completely replace magnesium. When the Tris concentration was increased (50 mM), spermidine could not replace magnesium. Supernatant and ribosomal fractions from T. thiooxidans were exchanged with those of Bacillus thuringiensis and Escherichia coli, and the ribosomal fraction from the chemolithotroph gave good to moderate stimulation when exchanged with the supernatant from the heterotrophs. On the other hand, the supernatant from T. thiooxidans gave good stimulation when mixed with ribosomes from B. thuringiensis but poor activity with ribosomes from E. coli. Both supernatant and ribosomal fractions prepared from stationary phase extracts of T. thiooxidans were inactive in the cell-free system.     

Mammalian mitochondrial ribosomes. Studies on the exchangeability of polypeptide chain elongation factors from bacterial and mitochondrial systems

Mammalian mitochondrial ribosomes from rat liver synthesised poly(phenylalanine) from [14C]-Phe-tRNA in the presence of a homologous 10(5) X gav supernatent fraction. The activity depended on the addition of synthetic template and was resistant to cycloheximide. The polyanion spermidine had a stimulatory effect on peptide synthesis in vitro. In contrast to Escherichia coli ribosomes, which also functioned with heterologous supernatant fractions, 55-S mitochondrial ribosomes were inactive when supplemented with heterologous supernatant fractions from E. coli or with purified bacterial elongation factors. EF-T slightly stimulated polyphenylalanine synthesis when added in combination with mitochondrial supernatant fractions. Two-dimensional electrophoretic analysis of the protein content of both supernatant fractions revealed considerable differences in the distribution of the species-specific proteins according to their isoelectric points. The mitochondrial supernatant proteins were in general more basic, and the few acidic proteins did not co-migrate with EF-Tu or EF-G from E. coli.     

Localization of polyamine enhancement of protein synthesis to subcellular components of Escherichia coli and Pseudomonas sp. strain Kim.

At 5 mM Mg2+, spermidine stimulation of polyphenylalanine synthesis by cell-free extracts of Escherichia coli was found to be about 30 times greater than that by extracts of Pseudomonas sp. strain Kim, a unique organism which lacks detectable levels of spermidine. By means of reconstitution experiments, the target of spermidine stimulation was localized to the protein fraction of the highspeed supernatant component (S-100) of E. coli and was absent from, or deficient in, the S-100 fraction of Pseudomonas sp. strain Kim. The spermidine stimulation did not appear to be due to the presence in the E. coli S-100 fraction of ribosomal protein S1, elongation factors, or E. coli aminoacyl-tRNA synthetases. The failure to observe spermidine stimulation by the Pseudomonas sp. strain Kim S-100 fraction was also not due to a spermidine-enhanced polyuridylic acid degradation. The synthesis of polyphenylalanine by Pseudomonas sp. strain Kim extracts was stimulated by putrescine and by S-(+)-2-hydroxyputrescine to a greater degree than was synthesis by E. coli extracts. The enhancement by putrescine and by S-(+)-2-hydroxyputrescine with Pseudomonas sp. strain Kim extracts was found to be due to effects on its ribosomes.     

Toxicity of chitinase-producing Bacillus thuringiensis ssp. kurstaki HD-1 (G) toward Plutella xylostella

One-hundred fifty isolates of Bacillus thuringiensis were tested for their ability to produce chitinase using colloidal chitin agar as the primary plating medium. Of 14 strains that produced chitinase, B. thuringiensis ssp. kurstaki HD-1(G) was identified as the highest chitinase producer and selected for further study. This bacterium produced the highest amount of chitinase (19.3 mU/ml) when it was cultivated in nutrient broth supplemented with 0.3% colloidal chitin on a rotary shaker (200 rpm) at 30 degrees C for 2 days. The toxicities of B. thuringiensis ssp. kurstaki HD-1(G) and B. thuringiensis ssp. kurstaki wa-p-2, a chitinase nonproducer, were assayed toward Plutella xylostella (diamondback moth) larvae, resulting in LC(50)'s of 4.93 x 10(4) and 1.32 x 10(5) spores/ml, respectively. If the culture broth from B. thuringiensis ssp. kurstaki HD-1(G) was used as the suspending liquid instead of phosphate buffer, their LC(50)'s were reduced to 6.23 x 10(3) and 7.60 x 10(4) spores/ml, respectively. The histopathological changes of the midgut epithelial cells of diamondback moth larvae were compared after feeding on B. thuringiensis ssp. kurstaki HD-1(G) with and without the presence of supernatant containing chitinase under light microscopy and transmission electron microscopy. The midgut epithelial cells of larvae fed for 30 min in the presence of chitinase, with or without spores and endotoxin crystals, appeared more elongated and swollen than those of the control larvae. A number of different cellular changes such as extensive cellular disintegration and appearance of numerous vacuoles were observed from the larvae fed on B. thuringiensis ssp. kurstaki HD-1(G) supplemented with supernatant containing chitinase. Thus increased toxicity and changes in epithelial cells were correlated with the presence of chitinase but this was not distinguished from the possible presence of vegetative-stage insecticidal proteins.     

Increase of fidelity of polypeptide synthesis by spermidine in eukaryotic cell-free systems

The mechanism of spermidine-induced increase of fidelity of polypeptide synthesis in a wheat germ cell-free system has been studied. It was found that the increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl-tRNA to ribosomes, that reduction of misreading was more marked at the 5'-base than at the 3'-base of the codon and that misreading caused by paromomycin and kanamycin C was not significantly decreased by spermidine. It was deduced from these results that spermidine inhibited low-frequency misreading more strongly than high-frequency misreading. In addition, spermidine was found to stimulate the rejection of non-cognate aminoacyl-tRNA mainly at an initial discrimination step during the binding of amino-acyl-tRNA to ribosomes, and slightly at a subsequent GTP-dependent discrimination step, the so-called proofreading step. In yeast, rabbit reticulocyte, and Artemia salina cell-free systems, spermidine was found to increase the fidelity of protein synthesis.     

Effect of polyamines on the stability of brain-cortex ribosomes

1. Ribosomes isolated from the cortex tissue of goat brain contain very small amounts of spermidine and spermine. Ribosomes isolated from spermidine-treated slices have a higher spermidine content. 2. The polyamines partially prevent the temperature-dependent breakdown of ribosomes into acid-soluble nucleotides. 3. The ;melting' temperature of ribosomes rises slightly when the ribosomes are heated slowly in the presence of polyamines. 4. The pH-dependent breakdown of ribosomes into protein, RNA and acid-soluble nucleotide is markedly decreased by polyamines present in media in which ribosomes are suspended. 5. The breakdown of ribosomes in the presence of high concentrations of salts and EDTA is partially checked by the concurrent presence of polyamines. 6. Spermidine and spermine make ribosomes less susceptible to enzymic digestion by crystalline trypsin and ribonuclease.     

A Cucumis sativus cell-free translation system: preparation, optimization and sensitivity to some antibiotics and ribosome inactivating proteins

A cell-free translation system was prepared from 3- to 5-day-old embryonic axes of gherkin ( Cucumis sativus L.). The system was optimized for Mg²⁺, K⁺, NH⁺₄, high speed supernatants, tRNA mixture from wheat germ, time and temperature. The system translates efficiently both endogenous mRNA (using a 30000 g supernatant) and polyuridylic acid (using either a 30000 g supernatant or a 100000 g supernatant supplemented with purified ribosomes). Translation by gherkin ribosomes was inhibited by several well-known eukaryotic inhibitors, antibiotics and ribosome-inactivating proteins. A translational inhibitory activity found in Cucumis sativus L. dry seeds acted on polypeptide synthesis carried out by cell-free systems from several mammals and plants, including gherkin embryonic axes. Our results indicate that the inhibitor is located in the seed bark and cotyledons, and is either blocked or absent in the embryonic axes, thus allowing the isolation of active gherkin ribosomes. The presence of the putative inhibitor appeared to be unevenly distributed in developing plants.     

Purification, amino acid sequence and characterization of Bacthuricin F4, a new bacteriocin produced by Bacillus thuringiensis

AIMS: Purification and characterization of a new bacteriocin, Bacthuricin F4 of Bacillus thuringiensis. METHODS AND RESULTS: A newly isolated B. thuringiensis subsp. kurstaki strain BUPM4, was shown to produce a novel bacteriocin named Bacthuricin F4. The highest bacteriocin activity was found in the growth medium and evidenced in the late exponential growth phase. Bacthuricin F4 could be purified by a two-step procedure: ammonium sulphate precipitation of protein from culture supernatant followed by a reverse phase chromatography. Upon purification, the specific activity was increased 100-fold. This bacteriocin was heat-stable up to 70 degrees C and resisted up to pH 3.0. Bacthuricin F4 was sensitive to proteases demonstrating its proteinaceous nature. Its molecular mass, determined by mass spectrometry was 3160.05 Da. Direct N-terminal sequencing of Bacthuricin F4 revealed the following sequence: DWTXWSXL. The latter was unique in the databases. Bacthuricin F4 was active against Bacillus species while it had little or no effect on Gram-negative bacteria. CONCLUSIONS: A strain BUPM4 of B. thuringiensis subsp. kurstaki, was shown to produce a new bacteriocin named Bacthuricin F4 of both new molecular mass (3160.05 Da) and new amino acid terminal sequence. This is, to our knowledge, the first bacteriocin exhibiting such characteristics reported to be produced by B. thuringiensis. SIGNIFICANCE AND IMPACT OF THE STUDY: The bacteriocin produced by the B. thuringiensis strain BUPM4 respond to both criteria of thermostability and stability to low pHs. Thus, it could be used for the control of the related species of Bacillus harmful for agricultural products.     

Factors involved in increased protein synthesis in rat kidney after administration of DDT

Homogenates of kidney cortex obtained from control rats and rats treated with DDT have been separated into microsomes or ribosomes, and into postmicrosomal (S105) supernatant fraction or pH 5 supernatant fraction. The incorporation of [¹⁴C]phenylalanyl-tRNA into peptide was increased when microsomes derived from kidneys of DDT-treated rats were incubated with pH 5 supernatant fraction from control rats. Elongation factors (EF) 1 and 2, necessary for the binding of aminoacyltRNA to ribosomes and for translocation of peptidyl-tRNA from the A site to the P site of ribosomes, were present in the pH 5 supernatant fractions of kidney of control and DDT-treated rats and these fractions were incubated with KCl-washed ribosomes obtained from livers of control rats. The results provided evidence that the increased incorporation observed with the pH 5 supernatant fraction obtained from the DDT-treated animals could not be attributed to decreased ribonuclease activity or to increased elongation factor 2 activity but was due to an increase in elongation factor l activity.     

Combined analysis of supernatant-based feeding bioassays and PCR as a first-tier screening strategy for Vip -derived activities in Bacillus thuringiensis strains effective against tropical fall armyworm

AIMS: To assess whether feeding bioassays using culture-supernatant proteins could be combined with PCR into a first-tier screening strategy for Vip3A-like genes efficient against tropical Spodoptera frugiperda. METHODS AND RESULTS: Out of 12 Bacillus thuringiensis strains studied, the total protein concentrated from the culture supernatant of only the strain HD125 yielded a significantly increased armyworm mortality and an intense band of the predicted size for VIP3A protein in SDS-PAGE. However, PCR and sequencing data indicated Vip-like genes are ubiquitous in tropical B. thuringiensis isolates. Interestingly, the HD125 strain was also the only one displaying a single-band amplification pattern and the highest sequence identity to the reported Vip3A(a) gene. CONCLUSIONS: Results suggest the insecticidal effectiveness of putative VIPs in B. thuringiensis isolates can be preliminarily estimated by the use of supernatant-derived total protein in feeding experiments, though only in a limited manner. SIGNIFICANCE AND IMPACT OF THE STUDY: A simple and cost-effective first-tier screening strategy for VIP-derived activities in B. thuringiensis collections can be developed by combining PCR and feeding bioassays. Moreover, the employed primers showed to be useful as a tool for strains differentiation at DNA level, and for characterization and isolation of Vip-like genes in tropical B. thuringiensis germplasm.     

Mescaline-induced changes of brain-cortex ribosomes. Role of spermidine in counteracting the destabilizing effect of mescaline on brain-cortex ribosomes

1. The effect of spermidine on the mescaline-induced changes of brain-cortex ribosomes was studied by adding spermidine during the treatment of goat brain-cortex slices with mescaline. 2. Mescaline treatment of brain-cortex slices removed a portion of the endogenous spermidine from ribosomes and this removal was significantly prevented when spermidine was present during mescaline treatment. 3. Spermidine present during mescaline treatment of brain-cortex slices counteracted, to some extent, the destabilizing effect of mescaline on ribosomes with respect to heat denaturation. 4. Mescaline treatment of brain-cortex slices made ribosomes more susceptible to breakdown, releasing protein and RNA, and resulting in loss of ribosomal enzymic activities. However, spermidine present during mescaline treatment counteracted moderately the mescaline-induced ribosomal susceptibility to breakdown and ribosomal loss of enzymic activities. 5. Ribosomes of mescaline-treated cortex slices were rapidly degraded by ribonuclease and trypsin. However, if spermidine was present during mescaline treatment of brain-cortex slices the rates of degradation diminished.     

Investigations on the association of phosphatidylserine synthase with the ribosomal component from Escherichia coli

The CDP-1,2-diacyl-sn-glycerol (CDP-diacylglycerol):L-serine O-phosphatidyltransferase (EC 2.7.8.8, phosphatidylserine synthase) of Escherichia coli is the first enzyme in the pathway committed to the biosynthesis of the major lipid in E. coli, phosphatidylethanolamine. The enzyme is unique among the phospholipid biosynthetic enzymes due to its high affinity for ribosomes in crude extracts. We report here investigations which define the nature of this in vitro affinity for ribosomes. Phosphatidylserine synthase can be dissociated from ribosomes in the presence of various inorganic salts at high ionic strength. Dissociation was also brought about by cellular levels of the polyamine spermidine. These results suggest the interaction of the enzyme with ribosomes in vitro is primarily ionic in nature, and polyamines may prevent this interaction in vivo. In the presence of nonionic detergent-lipid substrate mixed micelles under assay conditions the enzyme is also dissociated from ribosomes. Dissociation does not occur in the presence of detergent alone or in the presence of lipids which are not substrates or products of the enzyme. This dissociation by lipid substrate indicates the enzyme is not associated with ribosomes during catalysis.     

Regulation of protein synthesis in heat-shocked Drosophila cells. Soluble factors control translation in vitro

We have developed an in vitro translation system from heat-shocked and normal Drosophila cultured cells. The lysates retain regulation of translation typical of the whole cells from which they were prepared, both when programmed by endogenous mRNA and when RNA-dependent. These systems have been used to investigate the mechanism of shutdown of normal protein synthesis and selection of heat shock mRNAs for translation in heat shock in Drosophila. Supplementation of intact RNA-dependent lysates with separated ribosome or supernatant fractions from normal or heat-shocked translation systems showed the normal supernatant fraction could "rescue" normal protein synthesis in a heat shock lysate. Normal ribosomes had no rescuing activity and neither heat shock fraction affected translation in normal lysates. Reconstitution of the system from separated ribosomes and supernatant in normal and mixed combinations showed heat shock and normal ribosomes were both competent to support normal protein synthesis with normal supernatant. Heat shock supernatant did not support normal protein synthesis with ribosomes from either source. We conclude that the factors regulating translation in heat-shocked Drosophila cells are soluble factors in the lysate and that the soluble factors present in the normal lysate are dominant.     

Protein synthesis and the viability of rye grains. Loss of activity of protein-synthesizing systems in vitro associated with a loss of viability

A study was made of the integrity of some components of the protein-synthesizing system from viable and non-viable embryos of rye grains. In comparison with viable-embryo components both post-ribosomal supernatant and ribosomal fractions from non-viable embryos are impaired, for neither will fully support polyphenylalanine synthesis in poly(U)-directed cell-free systems. The lesion in the supernatant lies in components other than the tRNA or the aminoacyl-tRNA synthetase, for these are as functional as those present in the fully active cell-free systems from viable embryos. The ribosomes of embryos of lowered viability show considerable fragmentation and degradation of both 18S and 25S rRNA. This breakdown does not, however, account for the complete lack of polypeptide synthesis in the poly(U)-directed non-viable-embryo system, for if provided with viable-embryo supernatant, non-viable-embryo ribosomes will sustain 60% of the viable-embryo ribosome activity. A lesion in non-viable-embryo supernatant has been located in the binding of the aminoacyl-tRNA to the ribosome. The impaired components in both supernatant and ribosomes in systems in vitro may reflect the site of faults in protein synthesis in vivo in the early hours of germination. The development of these lesions during grain storage could contribute to senescence and loss of viability in the embryos of rye.     

苏云金芽胞杆菌肠毒素基因的PCR检测

采用多重引物PCR进行了 45株苏云金芽胞杆菌、2株蜡状芽胞杆菌和 2株球形芽胞杆菌溶血素BL ,肠毒素T和entS基因的检测 ,结果表明 95 6%苏云金芽胞杆菌含溶血素hblA基因 ,91 1 %含bceT基因 ,93 3%含entS基因。用两种商业化肠毒素检测试剂盒TECRA和RPLA进行所有菌株肠毒素的体外免疫测定 ,大部分苏云金芽胞杆菌和阳性蜡状芽胞杆菌都能产生不同水平的肠毒素活性 ,同hblA基因PCR检测结果基本相符。尽管DBT0 0 7和T2 4 0 0 1含有hblA基因 ,但用TECRA却检测不到肠毒素 ;Dmu39菌株不含肠毒素基因 ,但用TECRA却检测出高的肠毒素活性。苏云金芽胞杆菌BDT2 4 8和球性芽孢杆菌不含肠毒素基因和肠毒素。结果表明昆虫病原菌苏云金芽胞杆菌的安全性有待进一步研究     

Evidence for non-coordinated synthesis of 5 S RNA in the thermophilic fungus,Thermomyces lanuginosus

Analysis of ribosomes and the post ribosomal supernatant fraction of actively growing cells ofThermomyces lanuginosus showed the presence of free 5 S RNA in the supernatant fraction. This 5 S RNA was identical to the ribosomal 5 S RNA in its electrophoretic mobility on 10% Polyacrylamide gel and in its base composition. 5 S RNA from both the sources gave evidence for the presence of diphosphate at the 5’ end. Most of the 5 S RNA that appeared in the cytoplasm was that transported from the nucleus during the isolation. This could be prevented by the use of a hexylene glycol-HEPES buffer.     

The increase by spermidine of fidelity of protamine synthesis in a wheat-germ cell-free system

The influence of spermidine on the fidelity of natural mRNA-directed protein synthesis has been investigated. With protamine mRNA as a template for protamine synthesis, misincorporation of lysine, histidine, threonine and cysteine for arginine was measured in the presence and absence of spermidine. It was found that misincorporation of these four amino acids in the presence of spermidine was less than or nearly equal to that occurring in the absence of spermidine; however, incorporation of arginine was stimulated greatly by spermidine. These results clearly show that spermidine induced an increase of fidelity in protamine synthesis. The increase of fidelity in the presence of spermidine occurred mainly at the level of binding of aminoacyl-tRNA to ribosomes. The frequency of misreading the 5' base of the codon (misincorporation of cysteine) was greater than that of the middle base of the codon (misincorporation of histidine), but spermidine reduction of misreading was more marked at the middle base of the codon. Misincorporation of lysine (misreading of G to A residue at the middle base of the codon) was greater than that of threonine (misreading of G to C residue), but spermidine reduction of misreading was more marked in the misincorporation of threonine. It was deduced from these results that spermidine inhibited low-frequency misreading more effectively than high-frequency misreading.     

Properties of a revertant of Escherichia coli viable in the presence of spermidine accumulation: increase in L-glycerol 3-phosphate.

Escherichia coli CAG2242 cells are deficient in the speG gene encoding spermidine acetyltransferase. When these cells were cultured in the presence of 0.5 to 4 mM spermidine, their viability was greatly decreased through the inhibition of protein synthesis by overaccumulation of spermidine. When the cells were cultured with a high concentration of spermidine (4 mM), a revertant strain was obtained. We found that a 55-kDa protein, glycerol kinase, was overexpressed in the revertant and that synthesis of a ribosome modulation factor and the RNA polymerase sigma(38) subunit, factors important for cell viability, was increased in the revertant. Levels of L-glycerol 3-phosphate also increased in the revertant. Transformation of glpFK, which encodes a glycerol diffusion facilitator (glpF) and glycerol kinase (glpK), to E. coli CAG2242 partially prevented the cell death caused by accumulation of spermidine. It was also found that L-glycerol 3-phosphate inhibited spermidine binding to ribosomes and attenuated the inhibition of protein synthesis caused by high concentrations of spermidine. These results indicate that L-glycerol 3-phosphate reduces the binding of excess amounts of spermidine to ribosomes so that protein synthesis is recovered.     

Inhibition of the Peptide Bond Synthesizing Cycle by Chloramphenicol

To study the mechanism by which chloramphenicol inhibits bacterial protein synthesis, we examined the kinetics of the puromycin-induced release of peptides from transfer ribonucleic acid (tRNA) in the presence and in the absence of chloramphenicol. Washed Escherichia coli ribosomes with nascent peptides which had been radioactively labeled in vivo were used for this study. When such ribosomes were incubated in the presence of 10 mug of puromycin per ml, approximately one-fourth of the radioactive peptide material was rapidly released from tRNA. This rapid, puromycin-dependent reaction is assumed to be equivalent to the peptidyl transferase reaction. Chloramphenicol inhibited the extent of the puromycin-induced release of peptides by only 50%, demonstrating that some of the peptide chains which are present on active ribosomes react with puromycin, even in the presence of chloramphenicol. The addition of the supernatant fraction and guanosine triphosphate (GTP) increased the extent of the puromycin-induced release; this additional release was completely inhibited by chloramphenicol. Peptidyl chains on washed ribosomes prepared from chloramphenicol-inhibited cells were not released by puromycin in the presence of chloramphenicol and reacted slowly with puromycin in the absence of chloramphenicol. The release of peptidyl groups from these ribosomes became largely insensitive to chloramphenicol after preincubation of the ribosomes with GTP and the supernatant fraction. We conclude that chloramphenicol does not inhibit the peptidyl transferase reaction as measured by the puromycin-induced release of peptides from tRNA, but rather inhibits some step in the peptide synthesis cycle prior to this reaction.